The discovery of presenilins as ubiquitous intramembrane proteases in metazoans exemplifies an emerging trend in biology. The traditional distinction between basic and applied research has become increasingly blurred, as studies initiated with a strictly disease-oriented focus uncover fundamental biological mechanisms. In this renewal application, ten investigators who have collaborated successfully during the last decade on the normal and pathological biology of presenilin (PS) wish to extend their productive interactions into new experiments that will further illuminate the structure-function relationships of PS/ ? -secretase in normal biology and the role of this unusual multi-protein complex and various related gene products in the genesis of Alzheimer's disease. Based on extensive preliminary data in each of our four interrelated projects and supported by three vital cores, we will pursue numerous Specific Aims that include: 1. attempting to determine the structure of PS/ ?-secretase by performing cryo-electron microscopy of the purified, active complex as well as x-ray crystallography of the individual components; 2. extending our recent discovery that cholesterol dramatically enhances the catalytic activity of purified ? -secretase and that membrane lipids in general appear to represent the most potent regulators of both A production and Notch cleavage; 3. performing extensive genomic, functional genetic and protein analyses searching for novel lateonset AD candidate genes potentially implicated in presenilin-related pathways; 4. using advanced microscopy approaches in living cells (bimolecular fluorescence complementation; FLIM) to image and quantify the conformations and interactions of presenilin with APP, with new candidate genes emerging from (2), and with lipids and ? -modulating compounds identified herein; 5. identifying common binding partners and common subcellular processing pathways for the (32 subunit of NaJ, APP and APLP2, three proteins that are processed identically by both (3- and y-secretase. 6. performing an unbiased proteomics screen to more fully define the ? -secretome, i.e., the extent of unknown ?-substrates. These and numerous additional aims and sub-aims will be pursued collaboratively across our 4 projects, with progress in one project modifying experimental plans in another. In short, we are anxious to continue our close interaction to bring greater clarity to exactly how PS and its associated proteins and lipids process a host of substrates within membranes in health and disease. Lay summary: Ten experienced scientists who have collaborated productively for the past decade wish to address the structure and the functions of an unusual protein-cutting enzyme that is required for life in all multi-cellular animals and implicated in the causation of Alzheimer's disease.